Growing demand for alternative sources of energy can be fulfilled at least in part with a renewable supply of plant-derived biofuel oil and/or ethanol. To be a viable alternative to fossil fuels, the biofuel should provide a net energy gain in production, have environmental benefits, be economically competitive, and producible in large quantities without reducing food supplies, a current unintended byproduct of existing biofuel production.
The two predominant U.S. alternative transportation fuels, relative to fossil gasoline and diesel, are ethanol fermented from corn grain starch and biodiesel oil extracted from soybean seeds. Both corn and soybean are staple crops, on which national food supply significantly relies. Corn ethanol yields 25% more energy than the energy invested in its production, whereas biodiesel yields 93% more. Relative to the fossil fuels they displace, greenhouse gas emissions are reduced 12% by the production and combustion of ethanol and 41% by biodiesel. Biodiesel also releases fewer air pollutants per net energy gain than ethanol. These advantages of biodiesel over ethanol come from lower agricultural inputs and more efficient conversion of feedstocks to fuel. However, according to a recent estimation by Hill et al. (Environmental, economic, and energetic costs and benefits of biodiesel and ethanol biofuels. Proc Natl Acad Sci USA. 2006, 103(30):11206-11210), even dedicating all U.S. corn and soybean production to biofuels would meet only 12% of the gasoline demand and 6% of the demand for diesel.
Ideally, what is needed to support national alternative transportation fuel demands is biofuels produced from low-input biomass grown on agriculturally marginal land, using high-biomass plant species that are not involved in the food supply chain.
Growing demand for alternative sources of energy can be fulfilled with a renewable supply of plant-derived fuel oil and/or ethanol. Plants represent a significant source of biofuel vegetable oil because many species accumulate oil lipids as major storage components in seeds. The main form of vegetative storage oil in seeds, which represent, depending on the species, 15-50% of seed weight, is triacylglycerol (TAG). However, the primary substrate for oil synthesis are the carbohydrates generated in green photosynthetic tissues (leaves and stems) that are subsequently metabolized in chloroplasts to produce free fatty acids and acetyl-CoA units, the basic building blocks for TAG. Therefore, plant leaves are the main place of building block synthesis for TAG, and as it has been experimentally examined, the amount of TAG accumulated in oil seeds may be in part determined by the amount of fatty acid produced in plastids. (Bao X, Ohlrogge J. Supply of fatty acid is one limiting factor in the accumulation of triacylglycerol in developing embryos. Plant Physiol. 1999, 120:1057-62). Final storage of TAG occurs in seeds in small spherical organelles termed oil bodies (Heterogeneity of the endoplasmic reticulum with respect to lipid synthesis in developing seeds of Brassica napus L. Planta, 1996, 208:503-511; Wahlroos T, Soukka J, Denesyuk A, Wahlroos R, Korpela T, Kilby N J. Oleosin expression and trafficking during oil body biogenesis in tobacco leaf cells. Genesis. 2003, 35(2):125-132; Katavic V, Agrawal GK, Hajduch M, Harris SL, Thelen JJ. Protein and lipid composition analysis of oil bodies from two Brassica napus cultivars. Proteomics. 2006, 16: 4586-4598). Only about 0.2-0.3% of leaf biomass is represented by TAG.
With the advances in molecular biology and plant transformation technology, the metabolic engineering of fatty acids and vegetable oils has become possible (Gunstone FD, Pollard M. Vegetable oils with fatty acid changes by plant breeding or genetic modification. In F D Gunstone, ed, Structured and Modified Lipids. Marcel Dekker, New-York, pp 155-184 (2001); Thelen JJ, Ohlrogge JB. Metabolic engineering of fatty acid biosynthesis in plants. Metabolic Engineering 4, 12-21 (2002)). Plant oils represent some of the first successes in the design of improved plant products, and tobacco has been used as one of the first model plants to express genetically engineered fatty acids (Cahoon EB, Shanklin J, Ohlrogge JB. Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco. Proc Natl Acad Sci USA. 1992 89(23):11184-8; Reddy AS, Thomas TL. Expression of a cyanobacterial delta 6-desaturase gene results in gamma-linolenic acid production in transgenic plants. Nat Biotechnol. 1996, 14:639-642). The plant tissues that usually do not accumulate high amounts of oil still contain the enzymes used to synthesize these storage compounds.
High biomass plants, particularly broad leaf high biomass plants, have great biofuel potential. Plants that can yield between 100-400 tons/acre of low-cost, high-value biomass materials are particularly useful especially when there are none of the high costs, labor requirements, chemical inputs, or geographic restrictions associated with low biomass plant production.
While almost all plants have been investigated as alternative energy resources, tobacco (Nicotiana tabacum and other species from the Nicotiana genus) has been mostly overlooked. Similar to hardwood trees, tobacco will coppice or re-sprout from its stump after it has been cut. Coppicing makes multiple harvests in a year possible, enabling it to produce very high biomass tonnage. Tobacco thrives on different kinds of soil in a wide range of environments. The yield of tobacco seeds amounts to 600 kg/ha. The oil content in tobacco seed ranges between 36% and 41% by weight (Giannelos PN, Zannikos S, Stournas S, Lois E, Anastoloulos G. Tobacco seed oil as an alternative diesel fuel: physical and chemical properties. Industrial Crops and Products 2002, 16:1-9), indicating the existence of potent oil synthesis machinery, comparable to one of the traditional oil producers, such as soybean or rapeseed. Recent experiments indicated that tobacco seed oil can partially substitute petroleum diesel fuel at most operating conditions in terms of performance parameters and emissions without any engine modification and preheating of the blends (Gunstone F.D., Pollard M., Vegetable oils with fatty acid changes by plant breeding or genetic modification. In F.D. Gunstone, ed, Structured and Modified Lipids. Marcel Dekker, New-York, pp 155-184 (2001); Usta N. Use of tobacco seed oil methyl ester in a turbocharged indirect injection diesel engine. Biomass and Bioenergy 2005, 28: 77-86).
While the oil is accumulated at such high levels in seeds, (tobacco seed oil is used for some cosmetic and pharmaceutical needs), oil deposition in leaves is much lower, making the downstream oil extraction rather expensive. However, extraction of oil from leaf biomass might be cost-efficient in the case of significant improvement of oil content in leaves. This invention provides a technology for increasing oil yield from the green biomass of plants using an innovative biotechnology approach.